Protein splicing is a post-translational process catalyzed by a family of proteins known as inteins.(1) During this process, an intein domain catalyzes its own excision from a larger precursor protein and simultaneously ligates the two flanking polypeptide sequences (exteins) together. While most inteins catalyze splicing in cis, a small subset of these proteins exist as naturally fragmented domains that are separately expressed but rapidly associate and catalyze splicing in trans. Given their capacity to make and break polypeptide bonds (inteins can be considered protein ligases), both cis and trans-splicing inteins have found widespread use as chemical biological tools.(2)
Despite the growing use of inteins in chemical biology, their practical utility has been constrained by two common characteristics of the family, namely (i) slow kinetics and (ii) context dependent efficiency with respect to the immediate flanking extein sequences.(3,4) Recently, a split intein from the cyanobacterium Nostoc punctiforme (Npu) was shown to catalyze protein trans-splicing on the order of a minute, rather than hours like most cis- or trans-splicing inteins.(5) Furthermore, this intein was slightly more tolerant of sequence variation at the critical +2 C-extein residue than other inteins.(6)
Thus, a need exists for more robust and more efficient split inteins for use in a variety of protein purification and protein modification applications.